Turner J R, Angle J M, Black E D, Joyal J L, Sacks D B, Madara J L
Department of Pathology,Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Am J Physiol. 1999 Sep;277(3):C554-62. doi: 10.1152/ajpcell.1999.277.3.C554.
The mechanisms by which protein kinase C (PKC) activation results in increased transepithelial resistance (TER) are unknown [G. Hecht, B. Robinson, and A. Koutsouris. Am. J. Physiol. 266 (Gastrointest. Liver Physiol. 29): G214-G221, 1994]. We have previously shown that phosphorylation of the regulatory light chain of myosin II (MLC) is associated with decreases in TER and have suggested that contraction of the perijunctional actomyosin ring (PAMR) increases tight junction (TJ) permeability [J. R. Turner, B. K. Rill, S. L. Carlson, D. Carnes, R. Kerner, R. J. Mrsny, and J. L. Madara. Am. J. Physiol. 273 (Cell Physiol. 42): C1378-C1385, 1997]. We therefore hypothesized that PKC activation alters TER via relaxation of the PAMR. Activation of PKC by the phorbol ester phorbol 12-myristate 13-acetate (PMA) resulted in a progressive dose-dependent increase in TER that was apparent within 15 min (111% of controls) and maximal within 2 h (142% of controls). Similar increases were induced by a diacylglycerol analog, and the effects of both PMA and the diacylglycerol analog were prevented by the PKC inhibitor bisindolylmaleimide I. PMA treatment caused progressive decreases in MLC phosphorylation, by 12% at 15 min and 41% at 2 h. Phosphorylation of MLC kinase (MLCK) increased by 64% within 15 min of PMA treatment and was stable over 2 h (51% greater than that of controls). Thus increases in MLCK phosphorylation preceded decreases in MLC phosphorylation. These data suggest that PKC regulates TER via decreased phosphorylation of MLC, possibly due to inhibitory phosphorylation of MLCK. The decreased phosphorylation of MLC likely reduces PAMR tension, leading to decreased TJ permeability.
蛋白激酶C（PKC）激活导致跨上皮电阻（TER）增加的机制尚不清楚[G. 赫希特、B. 罗宾逊和A. 库苏里斯。《美国生理学杂志》266（胃肠肝脏生理学29）：G214 - G221，1994]。我们之前已经表明，肌球蛋白II调节轻链（MLC）的磷酸化与TER降低有关，并提出连接周肌动球蛋白环（PAMR）的收缩会增加紧密连接（TJ）的通透性[J. R. 特纳、B. K. 里尔、S. L. 卡尔森、D. 卡恩斯、R. 克纳、R. J. 姆斯尼和J. L. 马达拉。《美国生理学杂志》273（细胞生理学42）：C1378 - C1385，1997]。因此，我们假设PKC激活通过PAMR的松弛来改变TER。佛波酯佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯（PMA）激活PKC导致TER呈剂量依赖性逐渐增加，15分钟内明显增加（为对照的111%），2小时内达到最大值（为对照的142%）。二酰基甘油类似物也诱导了类似的增加，并且PKC抑制剂双吲哚基马来酰亚胺I可阻止PMA和二酰基甘油类似物的作用。PMA处理导致MLC磷酸化逐渐降低，15分钟时降低12%，2小时时降低41%。PKC激活剂PMA处理15分钟内，肌球蛋白轻链激酶（MLCK）的磷酸化增加64%，并在2小时内保持稳定（比对照高51%）。因此，MLCK磷酸化增加先于MLC磷酸化降低。这些数据表明，PKC通过降低MLC的磷酸化来调节TER，这可能是由于MLCK的抑制性磷酸化所致。MLC磷酸化的降低可能会降低PAMR张力，从而导致TJ通透性降低。
